The objective of this proposal is to analyze factors governing tolerance and autoreactivity among murine helper T (Th) and B cells in transgenic mice that express the influenza virus A/PR/8/34 hemagglutinin (PR8 HA) as a well-characterized neo-self antigen (HA Tg mice). The capacities for HA-specific Th and B cells that evade negative selection from the primary T and B cell repertoires in HA Tg mice to differentiate and participate in HA-specific immune responses will be examined. The following specific questions will be addressed: 1) What is the phenotype of HA-specific Th cells in HA Tg mice? HA Tg mice will be mated to transgenic mice expressing T cell receptors with defined specificities toward HA-derived class II-restricted neo-self peptides, and the extent and basis of negative selection of these HA-specific Th cells in HA Tg mice will be determined. Whether HA-specific Th cells from HA Tg and non-Tg (BALB/c) mice have differing capacities to differentiate into distinct Th phenotypes will be assessed. How Th phenotype (e.g., Th1, Th2, or autoreactive Th) affects the ability of HA-specific Th cells to provide help for humoral or cell-mediated immune responses will be evaluated. 2) What is the phenotype of HA-specific B cells in HA Tg mice? The extent to which HA-specific B cells that are activated following primary virus immunization are negatively selected during secondary B cell repertoire formation in HA Tg mice will be examined. HA Tg mice will be analyzed for the specificity of their secondary B cell responses to mutant viruses containing amino acid substitutions in B cell antigenic sites, to determine whether negative selection of PR8 HA-specific B cells focuses the secondary B cell responses of HA Tg mice toward mutant (non-self) epitopes and away from reactivity with the neo-self HA. Whether HA-specific B cells undergo somatic mutation and/or negative selection in the germinal center pathway in HA Tg mice will be assessed. The role that HA-specific B cells play in the activation of autoreactive Th cells will be evaluated by challenging HA Tg mice with isolated Th determinants versus the intact HA, for which HA-specific B cells can act as potent antigen presenting cells. 3) Does targeting expression to professional antigen presenting cells influence how the HA is recognized as a neo-self antigen? How expression of the HA in cell types that participate directly in immune repertoire formation affects the negative selection of autoreactive Th and B cells will be evaluated by analyzing Tg mice that express the HA under the control of a MHC class II promoter. Together, these studies will provide fundamental insights into the mechanisms of immune repertoire formation and tolerance. They will also have direct relevance to the processes of autoimmunity, since factors that determine the functional potential of autoreactive lymphocytes that evade negative selection and are activated by viruses bearing structural similarities to self antigens will be defined.